CN112703014A

CN112703014A - Treatment of EGFR mutant cancers

Info

Publication number: CN112703014A
Application number: CN201980060618.8A
Authority: CN
Inventors: A·哈塔; L·塞奎斯特; B·B·沃尔夫
Original assignee: Cable Chart Pharmaceutical Co; General Hospital Corp
Current assignee: Cable Chart Pharmaceutical Co; General Hospital Corp; Blueprint Medicines Corp
Priority date: 2018-08-10
Filing date: 2019-08-09
Publication date: 2021-04-23
Also published as: JP2021534129A; EP3833372A4; US20210308134A1; EP3833372A2; WO2020033838A3; WO2020033838A2

Abstract

Disclosed herein are methods for treating EGFR mutant cancers in a patient in need thereof by administering to the patient a therapeutically effective amount of at least one RET inhibitor (e.g., compound 1 and/or a pharmaceutically acceptable salt thereof) and a therapeutically effective amount of at least one EGFR inhibitor (e.g., ocitinib and/or a pharmaceutically acceptable salt thereof), and a combination therapy comprising at least one RET inhibitor and at least one EGFR inhibitor.

Description

Treatment of EGFR mutant cancers

This application claims priority and benefit from U.S. patent application No. 62/717,480 filed on day 10, 8, 2018 and U.S. patent application No. 62/735,730 filed on day 24, 9, 2018, each of which is incorporated herein by reference in its entirety.

The invention was made with government support under CA137008 and CA197389, issued by the National Institutes of Health. The government has certain rights in the invention.

The present disclosure relates to methods for treating EGFR mutant cancers in a patient in need thereof by administering a therapeutically effective amount of at least one RET inhibitor (e.g., at least one selective RET inhibitor) to the patient and administering a therapeutically effective amount of at least one EGFR inhibitor to the patient. For example, the present disclosure relates to methods for treating EGFR mutant cancers in patients who have been previously treated with at least one EGFR inhibitor and in some cases acquired resistance to at least one EGFR inhibitor. The present disclosure also relates to combination therapies comprising at least one RET inhibitor, e.g., at least one selective RET inhibitor and at least one EGFR inhibitor. In some embodiments, the at least one RET inhibitor is a selective RET inhibitor selected from compound 1 and pharmaceutically acceptable salts thereof. In some embodiments, the at least one EGFR inhibitor is selected from ocitinib (osimertinib) and pharmaceutically acceptable salts thereof.

Rearranged (RET) Receptor Tyrosine Kinases (RTKs) during transfection, as well as glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor- α (GFR α), are required for the development, maturation and maintenance of a variety of neurological, neuroendocrine and urogenital tissue types. However, there is increasing evidence that aberrant activation of RET is a key driver of tumor growth and proliferation in many solid tumors (Mulligan lm., nat. rev. cancer.14:173-186 (2014)).

Oncogenic RET rearrangements have been found in 1-2% of NSCLCs (Lipson, D. et al, nat. Med.18:382-384 (2012); Takeuchi, K. et al, nat. Med.18:378-381 (2012); Stransky, N. et al, nat. Commun.5:4846 (2014)). Oncogenic RET rearrangements produce constitutively active kinases that promote tumorigenesis. Like Anaplastic Lymphoma Kinase (ALK) and c-ROS Oncogene (ROS) 1-rearranged NSCLC, RET-rearranged NSCLC typically has adenocarcinoma histology (although occasionally squamous) and occurs in young, non-smoking patients. (1S,4R) -N- ((S) -1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -1-methoxy-4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) cyclohexanecarboxamide (Compound 1) described herein is a potent and selective inhibitor of RET kinase and oncogenic RET mutants. In cellular systems, compound 1 inhibits kinase activity of RET oncogenic mutants with low nanomolar potency. The in vivo dose-dependent antitumor efficacy of compound 1 has been demonstrated in several RET-driven models. Notably, in the first human trial, compound 1 induced a durable clinical response without significant off-target toxicity in NSCLC patients with RET alterations (subciah, v. et al Cancer Disc (online release 4/15 days 2018)). Compound 1 is currently being investigated for the treatment of patients with RET-driven malignancies, such as thyroid cancer, non-small cell lung cancer (NSCLC) and other advanced solid tumors.

RET fusions may also be involved in the case of some EGFR mutant cancers (see Schrock, a.b. et al, j.thorac. oncol.doi:10.1016/j.jtho.2018.05.027 (published online 6/5 th 2018)). Although certain EGFR inhibitors have been approved for the treatment of cancer, such as non-small cell lung cancer (ocitinib), a subset of patients who progress on receiving EGFR inhibitor treatment have acquired gene fusions that lead to acquired resistance. RET fusions (see Oxnard, G.R. et al, JAMA Oncology doi:10.1001/JAMA Oncology.2018.2969 (published online 8.2.8.2018)) and Karen L.Reckakakyamp et al, Analysis of Cell-Free DNA from 32,991Advanced cancer receptors novels Co-Occurring Activating RET alternatives and oncogeneic signalling Pathway Abstract at AACR Annual Meeting 2018(Apr,15,2018)), such as CCDC6-RET, most commonly occur in the event of "loss" of the previously recorded EGFR T790M gating mutation.

In EGFR mutant patients, EGFR TKI resistance promoted by RET fusion is similar to shunt-tracked resistance promoted by MET amplification. In the case of MET amplification, both preclinical and clinical evidence showed strong responses by inhibition of EGFR and MET (Engleman, J.A. et al, Science316:1039-43 (2007); Gainor, J.F. et al, J.Thorac.Oncol.11(7): e83-e85 (2016); Ahn, M. et al, J.Thorac.Oncol.12(11S2): pS1768 (2017)).

However, for many patients with EGFR TKI resistance, treatment options are very limited and in most cases, progression of the cancer is uncontrolled. Thus, although EGFR inhibitors including EGFR TKIs are effective as monotherapy or dual inhibitor therapy (containing MET inhibitors) in certain cancers, and RET inhibitors have potential in certain cancers, there is still a need for even more effective cancer treatment regimens.

Disclosure of Invention

The following disclosure describes methods for treating EGFR mutant cancers in a patient in need thereof by administering to the patient a therapeutically effective amount of at least one RET inhibitor and a therapeutically effective amount of at least one EGFR inhibitor. For example, in some embodiments, the patient has been previously treated with at least one EGFR inhibitor. In some embodiments, the patient gains resistance to at least one EGFR inhibitor.

Illustratively, in some embodiments, the at least one RET inhibitor is a selective RET inhibitor, e.g., compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, compound 1 is administered orally to the patient once daily. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered to the patient once daily is 200mg to 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered to the patient once daily is 200mg to 300mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

In some embodiments, the at least one RET inhibitor is selected from: almanib (alectinib), apatinib (apatinib), BOS172738(DS-5010), cabozantinib (cabozantinib) (XL184), dovitinib (dovitinib) (TKI258), GSK3179106, GSK3352589, lenvatinib (lenvatinib), LOXO-292, SL-1001, TPX-0046, nintedanib (nintedanib), ponatinib (ponatinib), sinatinib (sitatinib), sinatinib (sitovatinib) (MGCD516), sorafenib (sorafenib), sunitinib (sunitinib), regorafenib (BAY 73-4506), RXDX X-105, vandetanib (vandetanib), XL999, and pharmaceutically acceptable salts of any of the foregoing.

In some embodiments, the at least one RET inhibitor is selected from: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multivitinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, nintedanib, ponatinib, seratinib (MGCD516), sorafenib, sunitinib, regrafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

In some embodiments, the at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof. In some embodiments, ocitinib and/or at least one pharmaceutically acceptable salt thereof is administered orally to the patient once daily. In some embodiments, the therapeutically effective amount of the at least one EGFR inhibitor administered to the patient once daily is 80mg of ocitinib or a weight equivalent of a pharmaceutically acceptable salt thereof.

In some embodiments, the EGFR mutant cancer is characterized by at least one EGFR mutation selected from L858R, Δ ex19, T790M, C797S, and L792H. In addition, in some embodiments, the EGFR mutant cancer is characterized by at least one EGFR mutation selected from the group consisting of T790M, C797S, and L792H. In some embodiments, the EGFR mutant cancer is characterized by at least two EGFR mutations. In some embodiments, the EGFR mutant cancer is characterized by three EGFR mutations. In some embodiments, the EGFR mutant cancer is further characterized by at least one RET alteration, e.g., a CCDC6-RET fusion. In some embodiments, the EGFR mutant cancer is lung cancer, e.g., Small Cell Lung Cancer (SCLC) or non-small cell lung cancer (NSCLC).

The following disclosure also describes combination therapies comprising at least one RET inhibitor (e.g., at least one selective RET inhibitor, such as compound 1 and/or a pharmaceutically acceptable salt thereof) and at least one EGFR inhibitor (e.g., ocitinib and/or a pharmaceutically acceptable salt of any of the foregoing).

Treatment of patients (e.g., humans) with EGFR mutant cancers with at least one RET inhibitor in combination with at least one EGFR inhibitor can improve the treatment outcome of patients with EGFR TKI acquired resistance.

Exemplary embodiments of the present disclosure further include:

1. a method for treating an EGFR mutant cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of at least one RET inhibitor and a therapeutically effective amount of at least one EGFR inhibitor.

2. The method of embodiment 1, wherein at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof.

3. The method of embodiment 1, wherein at least one RET inhibitor is selected from the group consisting of: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multiverinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, Nintenib, Ponatinib, Serratinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

4. The method of embodiment 1, wherein at least one RET inhibitor is a selective RET inhibitor.

5. The method of any one of embodiments 1 to 4, wherein at least one EGFR inhibitor is a selective EGFR inhibitor.

6. The method of any one of embodiments 1 to 4, wherein at least one EGFR inhibitor is a third-generation EGFR inhibitor.

7. The method of any one of embodiments 1 to 4, wherein at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof.

8. The method of any one of embodiments 1 to 7, wherein the EGFR mutant cancer is characterized by at least one EGFR mutation selected from the group consisting of T790M, C797S, and L792H.

9. The method of any one of embodiments 1 to 8, wherein the EGFR mutant cancer is further characterized by at least one RET fusion.

10. The method of embodiment 9, wherein the EGFR mutant cancer is further characterized by a CCDC6-RET fusion.

11. The method of any one of embodiments 1 to 10, wherein the EGFR mutant cancer is lung cancer.

12. The method of embodiment 11, wherein the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.

13. The method of any one of embodiments 1 to 12, wherein the patient is a human.

14. The method of any one of embodiments 1 to 13, wherein the patient has been previously treated with at least one EGFR inhibitor.

15. The method of any one of embodiments 1 to 14, wherein the patient gains resistance to at least one EGFR inhibitor.

16. The method of any one of

embodiments

1, 2, and 4 to 15, wherein:

at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof;

at least one RET inhibitor is administered orally to a patient once daily; and is

The therapeutically effective amount of the at least one RET inhibitor is 200mg to 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

17. The method of embodiment 16, wherein the therapeutically effective amount of at least one RET inhibitor is 200mg to 300mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

18. The method of any one of embodiments 7 to 17, wherein

At least one EGFR inhibitor selected from ocitinib and pharmaceutically acceptable salts thereof;

orally administering at least one EGFR inhibitor to the patient once a day; and the therapeutically effective amount of the at least one EGFR inhibitor is 80mg of ocitinib or a weight equivalent of a pharmaceutically acceptable salt thereof.

19. A combination therapy comprising at least one RET inhibitor and at least one EGFR inhibitor.

20. The combination therapy of embodiment 19, wherein at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof.

21. The combination therapy of embodiment 19, wherein at least one RET inhibitor is selected from the group consisting of: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multiverinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, Nintenib, Ponatinib, Serratinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

22. The combination therapy of embodiment 19, wherein at least one RET inhibitor is a selective RET inhibitor.

23. The combination therapy of any one of embodiments 19-22, wherein at least one EGFR inhibitor is a selective EGFR inhibitor.

24. The combination therapy of any one of embodiments 19-22, wherein at least one EGFR inhibitor is a third-generation EGFR inhibitor.

25. The combination therapy of embodiment 20, wherein compound 1 is present in an amount of 200mg to 400 mg.

26. The combination therapy of embodiment 20, wherein compound 1 is present in an amount of 200mg to 300 mg.

27. The combination therapy of any one of embodiments 19 to 22, 25 or 26, wherein at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof.

28. The combination therapy of embodiment 27, wherein ocitinib is present in an amount of 80 mg.

29. A method for treating a patient having an EGFR mutant cancer, the method comprising:

(a) obtaining a biological sample from a patient;

(b) detecting the presence or absence of at least one RET fusion in a biological sample; and

(c) administering a combination therapy to the patient if at least one RET fusion is detected, wherein the combination therapy comprises at least one EGFR inhibitor and at least one RET inhibitor.

30. The method of embodiment 29, wherein at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof.

31. The method of embodiment 29, wherein at least one RET inhibitor is selected from the group consisting of: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multiverinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, SL-1001, TPX-0046, Nintenib, Ponatinib, Serratinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

32. The method of embodiment 29, wherein at least one RET inhibitor is a selective RET inhibitor.

33. The method of any one of embodiments 29 to 32, wherein at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof.

34. The method of any one of embodiments 29 to 32, wherein at least one EGFR inhibitor is a selective EGFR inhibitor.

35. The method of any one of embodiments 29 to 32, wherein at least one EGFR inhibitor is a third-generation EGFR inhibitor.

36. The method of any one of embodiments 29 to 35, wherein the EGFR mutant cancer is characterized by at least one EGFR mutation selected from the group consisting of T790M, C797S, and L792H.

37. The method of any one of embodiments 29 to 36, wherein at least one RET fusion is a CCDC6-RET fusion.

38. The method of any one of embodiments 29 to 37, wherein the EGFR mutant cancer is lung cancer.

39. The method of embodiment 38, wherein the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.

40. The method of any one of embodiments 29 to 39, wherein the patient is a human.

41. The method of any one of embodiments 29 to 40, wherein the patient has been previously treated with at least one EGFR inhibitor.

42. The method of any one of embodiments 29 to 41, wherein the patient gains resistance to at least one EGFR inhibitor.

The above embodiments are provided to introduce a selection of concepts discussed herein. These embodiments are not intended to identify essential features of the disclosed subject matter, or to limit the scope of the disclosed subject matter.

Drawings

Figure 1 is a scan showing that RECIST tumors shrink by 78% in a 60 year old female with EGFR del 19 and acquired CCDC6-RET fusions after two weeks of treatment with 200mg once daily compound 1 and 80mg once daily ocitinib, followed by six weeks of treatment with 300mg once daily compound 1 and 80mg once daily ocitinib. Serial coronal-to-enhancement computed tomography images of the chest revealed that right infrapulmonary masses and pleural nodules were seen at baseline (left) (arrows), with partial responses occurring eight weeks after treatment with compound 1 and ocitinib (right).

FIG. 2 shows CCDC6-RET expression cellsThe cell line model was generated by lentiviral infection of PC9(EGFR del 19) and MGH134(EGFR L858R/T790M) cells. From LC2/ad cells and PC9^CCDC6-RETOr MGH134^CCDC6-RETThe CCDC6-RET fusion gene or internal control TBP transcript of the cells was amplified by RT-PCR.

FIG. 3 shows PC9 in the presence or absence of Oscetinic acid^CCDC6-RETAnd MGH134^CCDC6-RETCell proliferation data of the cells. PC9 and MGH134 cells expressing CCDC6-RET gene fusion or Empty Vector (EV) were treated with 1 μ M ocitinib or Vehicle (VEH) and cell proliferation (ratio compared to the start of treatment) was determined within 5 days. Data shown are mean ± standard error of the mean (s.e.m.) of three independent biological replicates.

FIG. 4A is PC9 treated with 100nM Afatinib (Afatinib), 1 μ M Ositinib, Compound 1, or combinations thereof for 6 hours and harvested for Western blotting with antibody^EVAnd PC9^CCDC6-RETWestern blot of cells.

FIG. 4B is MGH134 treated with 1 μ M ocitinib, cabozantinib and Compound 1 or a combination thereof for 6 hours and harvested for Western blotting with the indicated antibodies^EVAnd MGH134^CCDC6-RETWestern blot of cells.

FIG. 4C shows PC9 treated with Compound 1 or with Afatinib or Oscetinib in the absence or presence of 1 μ M Compound 1^EVAnd PC9^CCDC6-RETCell viability of cells after 72 hours. For comparison purposes, the same compound 1 data is re-plotted in both figures. Data are shown as percentage of vehicle treated controls and are mean ± standard error of the mean of three independent biological replicates.

FIG. 5A is PC treated with 100nM Afatinib, 1 μ M ocitinib, cabozantinib, or combinations thereof for 6 hours and harvested for Western blot analysis^9VAnd PC9^CCDC6-RETWestern blot of cells.

FIG. 5B shows PC9 treated with cabozantinib or with afatinib or ocitinib in the absence or presence of 1 μ M Cabozantinib (CAB)^EVAnd PC9^CCDC6-RETCell viability of cells after 72 hours. For comparison purposes, the same cabozinib data are redrawn in both figures. Data are shown as percentage of vehicle treated controls and are mean ± standard error of the mean of three independent biological replicates.

FIG. 5C shows MGH134 treated with the RET inhibitor cabozantinib or Compound 1 or with oxcetinib in the absence or presence of 1 μ M RET inhibitor^EVAnd MGH134^CCDC6-RETCell viability of cells after 72 hours. Data are shown as percentage of vehicle treated controls and are mean ± standard error of the mean of three independent biological replicates.

Detailed description of the preferred embodiments

Abbreviations and Definitions

The following abbreviations and terms have the indicated meanings throughout:

as used herein, "combination therapy" refers to a therapy comprising more than one active agent. The two or more active agents may be administered in one dosage form or in separate dosage forms. In addition, the active agents comprising the combination therapy may be administered at the same time (in one or more dosage forms) or at separate times.

"Compound 1" is (1S,4R) -N- ((S) -1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -1-methoxy-4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) cyclohexanecarboxamide:

(Compound 1).

In 3 months 2017, compound 1 (also known as BLU-667 or paclitaxel) entered a phase I clinical trial (NCT03037385) in the united states for the treatment of patients with thyroid cancer, non-small cell lung cancer and other advanced solid tumors. WO 2017/079140, herein incorporated by reference, describes the synthesis of compound 1 (exemplary compound 130) and also discloses that this molecule inhibits, modulates and/or modulates the therapeutic activity of RET kinase (assay, example 10, pages 72-74).

As used herein, an "EGFR inhibitor" is a compound that inhibits EGFR kinase activity. The EGFR kinase is a wild-type EGFR kinase and/or one or more EGFR altered kinases (e.g., EGFR fusion, EGFR mutation, or EGFR copy number variation).

Examples of EGFR inhibitors include, but are not limited to: afatinib, ASP8273, avitinib (avitinib), bugatinib (brigitinib), cetuximab (cetuximab), dacomitinib (dacomitinib), EAI045, erlotinib (erlotinib), gefitinib (gefitinib), HS-10296, erlotinib (icotinib), lapatinib (lapatinib), rituximab (necitumumab), nazatinib (nazatinib), neratinib (neratinib), tematinib (olotinib), axitinib (olcetitinib), axitinib, panitumumab (panitumumab), PF-06747775, rocitinib (rociletinib) and vandetanib.

As used herein, a "fusion" is a protein resulting from a chromosomal translocation in which two genes are linked to an in-frame coding sequence and a chimeric protein is produced. In some embodiments, the fusion is a chromosomal translocation in which the kinase domain of one protein is fused to the dimerization domain of another gene.

As used herein, "RET fusion" is gene rearrangement. RET rearrangements produce fusion proteins that juxtapose the RET kinase domain with the dimerization domain of another protein, thereby producing constitutively active dimers that drive tumorigenesis.

As used herein, a "RET fusion protein" is the result of gene rearrangement. RET rearrangements produce fusion proteins that juxtapose the RET kinase domain with the dimerization domain of another protein, thereby producing constitutively active dimers that drive tumorigenesis.

As used herein, a "RET inhibitor" is a compound that inhibits RET kinase activity. The RET kinase is a wild-type RET kinase and/or one or more RET altered kinases (e.g., RET fusion, RET mutation, or RET copy number variation).

Examples of RET inhibitors include, but are not limited to, Alanib, Cabotinib (XL184), Compound 1, Duvitinib (TKI258), BOS172738(DS-5010), Forertinib (foretinib), Levatinib, LOXO-292, Prainitinib, RXDX-105, Serratinib (MGCD516), Sorafenib, sunitinib, TAS0286, TPX-0046, SL-1001, and vandetanib. Additional examples of RET inhibitors include, but are not limited to, apatinib, AUY-922, DCC-2157, GSK3179106, GSK3352589, motesanib (motesanib), nintendanib (nintendanib), NVP-AST487, PZ-1, regorafenib (BAY 73-4506), RPI-1, TG101209, SPP86, vatalanib (vatalanib), and XL 999.

Examples of RET inhibitors include, but are not limited to, Alanib, Cabotinib (XL184), Compound 1, Duvitinib (TKI258), BOS172738(DS-5010), Forertinib, Levatinib, LOXO-292, Prinertinib, RXDX-105, sersatinib (MGCD516), Sorafenib, sunitinib, TAS0286, and vandetanib. Additional examples of RET inhibitors include, but are not limited to, apatinib, AUY-922, DCC-2157, GSK3179106, GSK3352589, motesanib, nintedanib, NVP-AST487, PZ-1, regrafenib (BAY 73-4506), RPI-1, TG101209, SPP86, vartanib, and XL 999.

As used herein, a "selective RET inhibitor" refers to a compound or a pharmaceutically acceptable salt thereof that selectively inhibits RET kinase relative to another kinase and exhibits at least 2-fold selectivity for RET kinase relative to another kinase. For example, selective RET inhibitors exhibit at least 10-fold selectivity for RET kinase over another kinase; at least 15-fold selectivity; at least 20-fold selectivity; at least 30-fold selectivity; at least 40-fold selectivity; at least 50-fold selectivity; at least 60 times selective; at least 70-fold selectivity; at least 80-fold selectivity; at least 90-fold selectivity; at least 100-fold, at least 125-fold, at least 150-fold, at least 175-fold, or at least 200-fold selective. In some embodiments, the selective RET inhibitor exhibits at least 20-fold selectivity over another kinase (e.g., JAK 1). In some embodiments, the selective RET inhibitor exhibits at least 50-fold selectivity relative to another kinase (e.g., VEGFR-2 or TRKC). In some embodiments, the selective RET inhibitor exhibits at least 100-fold selectivity over another kinase (e.g., FLT3, JAK2, TRKA, or PDGFR β). In some embodiments, a selective RET inhibitor exhibits at least 1000-fold selectivity relative to another kinase (e.g., LIMK1, FGFR1, c-SRC, ML2/MAP3K10, PEAK1, FGFR3, MLK3/MAP3K11, ROS/ROS1, c-KIT, YES/YES1, FLT4/VEGFR3, JAK3, or TYK 2). In some embodiments, the selectivity for RET kinase over another kinase is measured in a cellular assay. In other embodiments, the selectivity for RET kinase over another kinase is measured in a biochemical assay.

Non-limiting examples of selective RET inhibitors include compounds I, SL-1001 and LOXO-292. Examples of selective RET inhibitors include compound 1 and LOXO-292.

As used herein, the term "subject" or "patient" refers to an organism to be treated by the methods of the present disclosure. Such organisms include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and in some embodiments, humans.

Many cancers have been associated with EGFR mutations. Such cancers are referred to herein as "EGFR mutant cancers". EGFR mutant cancers include lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, and lung squamous cell carcinoma), anal cancer, colon cancer, thyroid cancer (e.g., papillary thyroid cancer), glioblastoma, epithelial cancers (e.g., epithelial tumors of the head and neck). In some embodiments, the EGFR mutant cancer is characterized by at least one EGFR mutation selected from the group consisting of T790M, C797S, and L792H. In some embodiments, the EGFR mutant cancer is characterized by a T790M mutation. In some embodiments, the EGFR mutant cancer is characterized by a C797S mutation. In some embodiments, the EGFR mutant cancer is characterized by a L792H mutation. In some embodiments, the EGFR mutant cancer is characterized by an L858R or Δ ex19 mutation. In some embodiments, the EGFR mutant cancer is characterized by an L858R or Δ ex19 mutation and a T790M mutation. In some embodiments, the EGFR mutant cancer is characterized by an L858R or Δ ex19 mutation and a C796S mutation. In some embodiments, the EGFR mutant cancer is characterized by an L858R or Δ ex19 mutation, a T790M mutation, and a C796S mutation.

In some embodiments, the EGFR mutant cancer is further characterized by at least one RET fusion (e.g., at least one RET fusion listed in table 1). In some embodiments, the EGFR mutant cancer is further characterized by a CCDC6-RET fusion. In some embodiments, the EGFR mutant cancer is further characterized by KIF5B-RET fusion. In some embodiments, the EGFR mutant cancer is further characterized by NCOA4-RET fusion.

Table 1 RET fusions.

Some RET fusions in table 1 are discussed below: grubbs et al, J Clin Endocrinol Metab,100:788-93 (2015); halkova et al, Human Pathology 46:1962-69 (2015); U.S. patent nos. 9,297,011; U.S. patent nos. 9,216,172; le Rolle et al, Oncotarget 6(30):28929-37 (2015); antonescu et al, Am J Surg Pathol 39(7):957-67 (2015); U.S. patent application publication numbers 2015/0177246; U.S. patent application publication numbers 2015/0057335; japanese patent application publication No. 2015/109806 a; chinese patent application publication No. 105255927 a; fang et al, Journal of scientific Oncology 11.2(2016): S21-S22; european patent application publication No. EP3037547a 1; lee et al, Oncotarget DOI 10.18632/oncotarget.9137, pre-press electronic publishing 2016; saito et al, Cancer Science 107:713-20 (2016); pirker et al, Transl Lung Cancer Res,4(6):797-800 (2015); and Joung et al, Histopathology 69(1):45-53 (2016).

One of ordinary skill in the art can determine whether a subject has RET fusion, for example, using a method selected from the group consisting of: hybridization-based methods, amplification-based methods, microarray analysis, flow cytometry analysis, DNA sequencing, Next Generation Sequencing (NGS), primer extension, PCR, in situ hybridization, fluorescent in situ hybridization, dot blotting, and Southern blotting.

To detect fusion, a primary tumor sample can be collected from a subject. The sample is processed, the nucleic acids are isolated using techniques known in the art, and then sequenced using methods known in the art. The sequences are then mapped to individual exons and measures of transcriptional expression are quantified (such as RPKM, or mapped reads per million per kilobase read). Raw sequence and exon array data can be obtained from sources such as TCGA, ICGC, and NCBI gene expression integrated databases (GEO). For a given sample, individual exon coordinates are annotated with gene identifier information and exons belonging to the kinase domain are labeled. Exon levels were then normalized for z-score in all tumor samples.

Next, genes expressing the 5 'exon at levels significantly different from the 3' exon were identified. The sliding frame is used to identify breakpoints in individual samples. Specifically, at each iteration, incremental breakpoints separate the gene into 5 'and 3' regions, and the t statistic is used to measure the difference in expression (if any) between the two regions. The breakpoint with the largest t statistic is selected as the possible fused breakpoint. As used herein, a "breakpoint" is a boundary that fuses two different genes. This is sometimes referred to as a "fusion point". The position between 5 'and 3' where the difference in exon expression is greatest is the putative fusion breakpoint. Thousands of tumor samples can be analyzed rapidly in this manner, generating a fusion candidate list (ordered by t statistic). The advanced candidates can then be validated and fusion partners identified by examining the original RNA-seq dataset and identifying chimeric pairs and/or split reads that support fusion. Candidate fusions can then be confirmed experimentally as described below.

Alternatively, fusion can be identified by circulating tumor dna (ctdna) analysis of plasma (i.e., liquid biopsy).

In addition, described in Wang L et al, Genes Chromosomees Cancer 51(2):127-39(2012). doi 10.1002/gcc.20937, electronically published on 27/10/2011; and Suehara Y et al, Clin Cancer Res.18(24):6599-608 (2012): doi:10.1158/1078-0432.CCR-12-0838, methods electronically published in 10.10.2012 can also be used to detect fusions.

In some embodiments of the disclosure, the EGFR mutant cancer is lung cancer. In some embodiments, the EGFR mutant cancer is small cell lung cancer. In some embodiments, the EGFR mutant cancer is non-small cell lung cancer. In some embodiments, the EGFR mutant cancer is lung squamous cell carcinoma.

In some embodiments, the EGFR mutant cancer is an anal cancer.

In some embodiments, the EGFR mutant cancer is colon cancer.

In some embodiments, the EGFR mutant cancer is thyroid cancer. In some embodiments, the EGFR mutant cancer is papillary thyroid cancer.

In some embodiments, the EGFR mutant cancer is a glioblastoma.

In some embodiments, the EGFR mutant cancer is an epithelial cancer. In some embodiments, the EGFR mutant cancer is an epithelial tumor of the head or neck.

In some embodiments, a patient having an EGFR mutant cancer has been previously treated with at least one EGFR inhibitor (e.g., ocitinib and/or a pharmaceutically acceptable salt thereof). In some embodiments, a patient having an EGFR mutant cancer acquires resistance to at least one EGFR inhibitor (e.g., ocitinib and/or a pharmaceutically acceptable salt thereof).

As used herein, the phrase "therapeutically effective amount" refers to an amount of active agent (e.g., compound 1 or a pharmaceutically acceptable salt thereof) sufficient to achieve a beneficial or desired result. A therapeutically effective amount may be administered in one or more administrations, applications or administrations, and is not intended to be limited to a particular formulation or route of administration.

As used herein, the phrase "weight equivalents of a pharmaceutically acceptable salt thereof" for a particular compound includes the weight of both the compound and the related salt. For example,

the tablets contain 47.7mg or 95.4mg of ocitinib mesylate, which is an ocitinib with a weight equivalent of 40mg or 80mg, respectively.

As used herein, the phrase "pharmaceutically acceptable salt thereof," if used in connection with an active agent distributed in a salt form, refers to any pharmaceutically acceptable salt form of the active agent. For example, pharmaceutically acceptable salts of oseltamiib mesylate include oseltamiib besylate, oseltamiib hydrochloride, and the like.

As used herein, the term "treating" includes any effect, e.g., reduction, modulation, or elimination, that results in the amelioration of a condition, disease, disorder, etc., or amelioration of a symptom thereof.

RET inhibitors that may be used in some embodiments include those well known in the art, for example, Alanib, Apatinib, AUY-922, Cabovatinib (XL184), Compound 1, DCC-2157, Multivitinib (TKI258), BOS172738(DS-5010), Foruitinib, GSK3179106, GSK3352589, Levatinib, LOXO-292, TPX-0046, SL-1001, Motifloxanib, Nidanib, NVP-487, Prainitinib, PZ-1, Regenafenib (BAY 73-4506), RPI-1, RXDX-105, TG101209, serrtinib (MGCD516), fesonib, sunitinib, RPI-1, TAS0286, TG101209, SPP86, Tatalanib, Vantanib, and PCT 999 as disclosed in the following publications: WO 2005/062795, WO 2007/087245, WO 2009/003136, WO 2009/100536, WO 2010/006432, WO 2014/039971, WO 2014/050781, WO 2014/141187, WO 2015/006875, WO 2015/079251, WO 2016/037578, WO 2016/038552, WO 2016/075224, WO 2016/127074, WO 2017/011776, WO 2017/079140, WO 2017/145050, WO 2017/161269, WO 2017/178844, WO 2017/178845, WO 2018/017983, WO 2018/022761, WO2018/064852, W02018/060714, WO 2018/071454, WO 2018/071447, WO2018/102455, WO2018/136661, WO 2018/136663, WO2018/189553, WO2018/136661, W20119/001556, WO2019/008172, WO2019/126121, WO2019/143977, WO2019/143991 and WO 2019/143994.

In some embodiments, the RET inhibitor is a multi-kinase inhibitor originally designed to target other kinases, such as vascular endothelial growth factor receptor 2(VEGFR-2), tyrosine protein kinase MET, and/or EGFR, which inhibits the other kinases more effectively than RET, such as cabozantinib, vandetanib, sunitinib, lenvatinib (levatinib), regorafenib, and RXDX-105. In some embodiments, the multi-kinase inhibitor with RET activity is a weak inhibitor of RET with a gating mutation at residue V804, such as V804L and V804M.

In some embodiments, at least one RET inhibitor is a selective RET inhibitor. In some embodiments, selective inhibitors are designed to efficiently and selectively target wild-type (WT) RET and oncogenic mutant forms of RET, such as common RET alterations, including RET fusions (e.g., KIF5B-RET, CCDC6-RET) and RET activation mutations (e.g., C634W, M918T, V804L/M), while maintaining selectivity for other human kinases. In some embodiments, the selective RET inhibitor has activity against multiple oncogenic mutant forms of RET, regardless of tumor type. In some embodiments, the equivalent activity of a selective RET inhibitor across multiple oncogenic mutant forms of RET distinguishes selective RET inhibitors from multi-kinase inhibitors with RET inhibitory activity.

In some embodiments, at least one RET inhibitor is a selective RET inhibitor. Compound 1 is a selective inhibitor of RET that inhibits only wild-type RET and one or more mutant forms of RET, and has little inhibitory activity against other kinases. LOXO-292 (Seerpatinib) is also a selective RET inhibitor. Selective RET inhibitors that may be used in some embodiments include those well known in the art, such as the compounds disclosed in: WO 2016/127074, WO 2017/011776, WO 2017/079140, WO 2017/161269, WO 2018/017983, WO 2018/022761, WO 2018/071454, WO 2018/071447, WO2018/136661, WO 2018/136663, WO2018/237134, W02019/001556, WO2019/143994, WO2019/143991 and WO 2019/143977.

For example, in some embodiments, the at least one RET inhibitor is a selective RET inhibitor selected from the group consisting of:

and a pharmaceutically acceptable salt of any of the foregoing.

In some embodiments, the at least one RET inhibitor is a selective RET inhibitor selected from the group consisting of: 4- (6- (4-benzylpiperazin-1-yl) pyridin-3-yl) -6- (2-morpholinoethoxy) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2-hydroxyethoxy) -4- (6- (6- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; (R) -6- (2-hydroxypropoxy) -4- (6- (4- ((6-methoxypyridin-3-yl) methyl) piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2-hydroxy-2-methylpropoxy) -4- (6- (6- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2-methoxyethoxy) -4-6- (4- ((6-methoxypyridin-3-yl) methyl) piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2-hydroxy-2-methylpropoxy) -4- (6- (6- (6-methoxynicotinoyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2- (dimethylamino) ethoxy) -4- (6- (6- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 4- (6- (6- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyridin-3-yl) -6- (2-morpholinoethoxy) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 4- (6- (6- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyridin-3-yl) -6- ((1-methyl-1H-imidazol-4-yl) methoxy) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6-ethoxy-4- (5- (6- ((5-fluoro-6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] hept-3-yl) pyrazin-2-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; and a pharmaceutically acceptable salt of any of the foregoing.

In some embodiments, the at least one RET inhibitor is a selective RET inhibitor selected from the group consisting of: n- (1- (5- (3-cyano-6- (2-hydroxy-2-methylpropoxy) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -4-methylpiperidin-4-yl) benzamide; 6-ethoxy-4- (6- (4-hydroxy-4- (pyridin-2-ylmethyl) piperidin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2-hydroxy-2-methylpropoxy) -4- (6- (3- (pyridin-2-yloxy) azetidin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 6- (2-hydroxy-2-methylpropoxy) -4- (6- (4- ((6-methoxypyridazin-3-yl) oxy) piperidin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; (S) -6- (2-hydroxy-2-methylpropoxy) -4- (6- (3- (pyridin-2-yloxy) pyrrolidin-1-yl) pyridin-3-yl) pyrazolo [ l,5-a ] pyridine-3-carbonitrile; n- (l- (5- (3-cyano-6- ((3-fluoro-1-methylazetidin-3-yl) methoxy) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -4-methylpiperidin-4-yl) -5-fluoro-2-methylbenzamide; 3-chloro-N- (1- (5- (3-cyano-6- ((3-fluoro-1-methylazetidin-3-yl) methoxy) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -4-methylpiperidin-4-yl) pyridinecarboxamide; n- ((3S,4S) -l- (5- (3-cyano-6-ethoxypyrazolo [ l,5-a ] pyridin-4-yl) pyridin-2-yl) -3-hydroxypiperidin-4-yl) -3-methylbutanamide; 6- (2-hydroxy-2-methylpropoxy) -4- (6- (4-hydroxy-4- (pyridin-2-ylmethyl) piperidin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile; 3-chloro-N- ((3S,4S) -l- (5- (3-cyano-6-ethoxypyrazolo [ l,5-a ] pyridin-4-yl) pyrazin-2-yl) -3-hydroxypiperidin-4-yl) pyridinecarboxamide; and a pharmaceutically acceptable salt of any of the foregoing.

In some embodiments, the at least one RET inhibitor is administered once daily. In some embodiments, at least one RET inhibitor is administered orally. In some embodiments, the at least one RET inhibitor is administered orally once daily.

In some embodiments, the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg to 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, or 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

In some embodiments, the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg to 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

In some embodiments, the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg to 300mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, or 300mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 200mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 205mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 210mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 215mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 220mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 225mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 230mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 235mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 240mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 245mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 250mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 255mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 260mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 265mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 270mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 275mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 280mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 285mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 290mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 295mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 300mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 305mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 310mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 315mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 320mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 325mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 330mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 335mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 340mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 345mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 350mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of at least one RET inhibitor administered orally once daily is 355mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 360mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 365mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 370mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 375mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 380mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 385mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 390mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 395mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount of the at least one RET inhibitor administered orally once daily is 400mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

Additionally, in some embodiments, the at least one EGFR inhibitor is selected from afatinib, ASP8273, ibrutinib, bugatinib, cetuximab, dacomitinib, EAI045, erlotinib, gefitinib, HS-10296, erlotinib, lapatinib, tolituzumab, azatinib, neratinib, temotinib, axitinib, panitumumab, PF-06747775, EGF816, YH5448, elvitinib, rocitinib, vandetanib, and pharmaceutically acceptable salts of any of the foregoing.

In some embodiments, the at least one EGFR inhibitor is a selective inhibitor. In some embodiments, the selective EGFR inhibitor is designed to efficiently and selectively target oncogenic mutant forms of EGFR, e.g., exon 19 deletion, L858R, T790M. In some embodiments, the selective EGFR inhibitor has activity against multiple oncogenic mutant forms of EGFR regardless of tumor type. In some embodiments, the selective EGFR inhibitor distinguishes the selective EGFR inhibitor from a multi-kinase inhibitor having EGFR inhibitory activity across the equivalent activity of multiple oncogenic mutant forms of EGFR.

In some embodiments, the selective EGFR inhibitor is a third-generation EGFR inhibitor. In some embodiments, the selective EGFR inhibitor has activity against oncogenic mutant forms of EGFR (including exon 19 deletion, L858R, and T790M). In some embodiments, the selective EGFR inhibitor comprises axitinib, rocitinib, imatinib, EGF816, PF-06747775, YH5448, and elvitinib. In some embodiments, the selective EGFR inhibitor does not have activity against WT EGFR. In some embodiments, the selective EGFR inhibitor is not a covalent inhibitor.

In some embodiments, the at least one EGFR inhibitor is administered once daily. In some embodiments, the at least one EGFR inhibitor is administered orally. In some embodiments, the at least one EGFR inhibitor is administered orally once daily.

In some embodiments, the at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof. In some embodiments, the therapeutically effective amount of the at least one EGFR inhibitor administered orally once daily is 80mg of ocitinib or a weight equivalent of a pharmaceutically acceptable salt thereof.

While the active agent (e.g., compound 1 or ocitinib) may be administered alone, in some embodiments, the active agent may be administered as a pharmaceutical formulation, wherein the active agent is combined with one or more pharmaceutically acceptable excipients or carriers. For example, the active agent may be formulated for administration in any convenient manner that is advantageous to human or veterinary medicine. In certain embodiments, the compounds included in the pharmaceutical formulations may be active themselves, or may be prodrugs capable of being converted to active compounds, for example, under physiological circumstances.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Examples of pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus membranaceus gel powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diol, sSuch as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) no pyrogen water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) a phosphate buffer solution; (21) cyclodextrins, e.g.

And (22) other non-toxic compatible ingredients employed in the pharmaceutical formulation.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Solid dosage forms (e.g., capsules, tablets, pills, dragees, powders, granules, and the like) can include one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binding agents, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerin; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant.

Liquid dosage forms may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspending agents, as for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Ointments, pastes, creams and gels may contain, in addition to the active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons (such as butane and propane).

Dosage forms for topical or transdermal administration of compound 1 include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

When compound 1 is administered as a medicament to humans and animals, it can be administered as such or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (such as 0.5% to 90%) of the active ingredient and a pharmaceutically acceptable carrier.

The formulations can be administered topically, orally, transdermally, rectally, vaginally, parenterally, intranasally, intrapulmonary, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intravesicularly, intradermally, intraperitoneally, subcutaneously, subcuticularly, or by inhalation.

The disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference.

Examples

Reagents and antibodies

Afatinib, ocitinib and cabozantinib were purchased from seleck Chemicals and resuspended in DMSO. Phosphorylated EGFR (Y1068), EGFR, pBRAF (Ser445), RET, pAKT (Ser473), AKT, pMEK1/2(Ser217/221), MEK1/2, pERK1/2(Thr202/204), ERK1/2, and actin antibodies were purchased from Cell Signaling Technology. The pRET (Y1062) antibody was obtained from Abcam.

RT-PCR and sequencing

Total RNA from cell lines was extracted using the RNeasy Mini Kit (Qiagen). SuperScript was used according to the manufacturer's instructions^TMII reverse transcriptase (Invitrogen) reverse transcribes RNA (1. mu.g). The following primers were used for PCR amplification of CCDC6-RET, PCBP2-BRAF and TBP: CCDC6 exons 1F-CGGACAGCGCCAGCG, RET exon 19R-GCATTATTACAGTCCACCAGCG; (PCBP2-BRAF primer 1) PCBP2 exon 6F-AGGTGGATGCAAGATCAAGG, BRAF exon 13R-TAGCCAGTTGTGGCTTTGTG; (PCBP2-BRAF primer 2) PCBP2 exon 2F-CGGTGTGATTGAAGGTGGAT, BRAF exon 18R-ACAGGAAACGCACCATATCC; TBP F-CCCATGACTCCCATGACC, TBP R-TTTACAACCAAGATTCACTGTGG. The PCR product was confirmed by agarose gel electrophoresis. After amplification, Sanger sequencing was performed.

Cell culture

PC9 and MGH134 cell lines are known in the art (Hata, A.N. et al, nat. Med.22(3):262-69(2016)), and cells were cultured in RPMI1640(Life Technologies) supplemented with 10% FBS. MGH845-1 cells were additionally cultured in 150nM ocitinib. All cells were routinely tested and confirmed to be free of mycoplasma contamination.

Generation of CCDC6-RET expressing cell lines

The CCDC6-RET fusion construct was synthesized by GenScript and ligated into the pLENTI6/V5-D-TOPO vector using the ViraPower lentivirus-directed TOPO expression kit (Life Technologies). Lentiviruses were generated by transfecting the pLENTI6 construct and packaging the plasmid into 293FT cells (Life Technologies). The virus production, collection and infection were all done according to the manufacturer's protocol. The transduced cells were selected in blasticidin (10mg/mL-20mg/mL) for one week.

Cell viability assay

For drug dose response assays, cells were seeded into 96-well plates 24 hours prior to drug addition. Cell proliferation was determined by the CellTiter-Glo assay (Promega) 72-120 hours after drug addition using standard protocols. For time course experiments, multiple plates were inoculated and dosed in the same manner, and plates were frozen at-80 ℃ at the indicated time points; all plates in the experiment were incubated with CellTiter-Glo at the same time. Luminescence was measured with a SpectraMax i3x multimode microplate reader (Molecular Devices).

Example 1: oscetinib AR biopsy

To better characterize the obtained resistance to ocitinib (AR), a single-center cohort of ocitinib AR biopsies was performed. Under IRB approved protocols, osetinib AR biopsies were performed by SNaPshot or Foundation One Next Generation Sequencing (NGS) and plasma was assayed by Guardant360 NGS. Specifically, 41 EGFR mutant patients treated with ocitinib for T790M + disease were queried by tissue NGS (n ═ 22), plasma NGS (n ═ 9), or both (n ═ 10). In 2 out of 32 tissue samples, SCLC conversion was observed. In 5 tissue samples and 5 plasma samples (all T790M were cis), EGFR C797S was found. In addition, MET amplification was observed in 7 tissues and 3 plasma samples. BRAF rearrangement was found in 2 of 32 tissue samples, while CCDC6-RET rearrangement was found in 1 of 32 tissue samples and 1 of 19 plasma samples. Tissue and plasma samples showing CCDC6-RET rearrangement were from different donors, suggesting that RET rearrangement is a low frequency but recurrent observation in EGFR mutant patients with AR to ocitinib.

Practice ofExample 2: study of patients

A 60 year old female with del 19 EGFR mutant advanced NSCLC received first line afatinib (one year), received T790M and was treated with ocitinib (18 months). She then underwent a lung biopsy showing CCDC6-RET fusion by SFA. The baseline tissue was not sufficient for Solid Fusion Assay (SFA), but RET Fluorescence In Situ Hybridization (FISH) was negative, indicating that CCDC6-RET fusion was obtained. The patients were written for an individual patient study new drug (IND) regimen for treatment with oxcetinca compound 1. She started 80mg oxcetinic acid per day and 200mg compound 1 per day and then increased compound 1 to 300mg 2 weeks after treatment. Within a few days after the start of treatment, her dyspnea improved. Scans after 8 weeks showed significant responses with 78% shrinkage of RECIST tumors (figure 1). The combination was well tolerated with only grade 1 toxicity, including fatigue, leukopenia, hypertension, dry mouth and elevated transaminases. Treatment was still ongoing by 2018, 9 and 24 months.

Bronchoscopic biopsy of a 44 year old male with del 19 EGFR mutant advanced NSCLC receiving first line cisplatin/pemetrexed, second line afatinib (one year) experienced increased lung lesions showing CCDC6-RET fusion by SFA. The baseline tissue is not available for RET testing. He received 150mg of erlotinib per day in combination with 60mg of labeled exocaptinib per day. Scans after one month showed stable disease (RECIST 1.1), but subsequent scans after 2.5 months showed disease progression and suggested treatment discontinuation. The patient had grade 1 diarrhea, rash and AST elevation

Example 3: expression of CCDC6-RET in EGFR mutant NSCLC cell lines confers resistance to EGFR inhibitors.

To determine whether CCDC6-RET expression was sufficient to cause acquired resistance, a cell line model expressing CCDC6-RET fusion was generated by lentivirus infection of PC9(EGFR del 19) and MGH134(EGFR L858R/T790M) cells (fig. 2).

In the absence of EGFR inhibitors, CCDC6-RET expressing cells grew similarly to the parental cells. When treated with Ocitinib (OSI), as compared to parental cells (EV) which experienced a net decrease in cell viability，PC9^CCDC6-RETAnd MGH134^CCDC6-RETThe cells continued to proliferate (FIG. 3). In the case of ocitinib treatment, the proliferation rate of CCDC6-RET expressing cells was reduced, indicating that RET activation, while sufficient to drive acquired resistance, did not fully compensate for EGFR signaling loss.

The effect of CCDC6-RET expression on downstream signaling pathway activation in PC9 and MGH134 cells was also examined. In PC9, in contrast to parental cells that do not express detectable RET protein^CCDC6-RETAnd MGH134^CCDC6-RETPhosphorylated RET was detected in both cells (fig. 4A-4B), CCDC6-RET expression alone did not result in an increase in downstream MAPK (phosphorylated ERK1/2) or PI3K (phosphorylated AKT) signaling activation at baseline; however, at PC9^CCDC6-RETAnd MGH134^CCDC6-RETIn cells, RET, ERK1/2, and AKT phosphorylation were retained in the presence of afatinib or ocitinib (fig. 4A-4B). Thus, expression of CCDC6-RET fusion may confer resistance to EGFR inhibitors in EGFR mutant NSCLC.

Example 4: acquired resistance caused by CCDC6-RET expression can be overcome by EGFR plus RET.

PC9CCDC6-RET cells produced as above were treated with compound 1 in the absence or presence of EGFR inhibitors. Treatment with compound 1 alone inhibited RET phosphorylation, but did not decrease downstream ERK or AKT phosphorylation (fig. 4A). Treatment with compound 1 in combination with either axitinib or afatinib completely inhibited both phosphorylated ERK and phosphorylated AKT and reduced cell viability to levels similar to parental cells treated with EGFR TKI (fig. 4C). At MGH134^CCDC6-RETSimilar results were observed in cells (fig. 4B, fig. 5C). In addition, PC9^CCDC6-RETAnd MGH134^CCDC6-RETCells were sensitive to EGFR TKI + cabozantinib (multi-kinase inhibitor with RET activity) (fig. 4B, fig. 5A to fig. 5C). Taken together, these data indicate that acquired resistance generated by CCDC6-RET fusion can be overcome by dual EGFR plus RET blockade.

Example 5: use of compound 1 and ocitinib for RET fusion mediated resistance to EGFR inhibition metastatic non-small cell lung cancerStudy (Combined study of metastatic NSCLC with RET-mediated resistance to EGFR inhibition)

This study is an open-label 1/2 phase study designed to evaluate the safety, tolerability, antitumor activity, PK and pharmacodynamics of the combination of the potent and selective RET inhibitor compound 1 with the third generation EGFR inhibitor oxcetinib in NSCLC patients who have developed RET fusions associated with oxcetinib resistance.

A dose escalation study was performed to assess the safety and tolerability of the combination of compound 1 with ocitinib and to determine the Maximum Tolerated Dose (MTD) and/or the recommended phase 2 dose (RP 2D). The overall safety profile of compound 1 in combination with ocitinib treatment was assessed by the type, frequency, severity, time and relationship to study drug, severe adverse events, vital sign changes, ECG and safety laboratory tests of any adverse event.

The study also estimated the Overall Response Rate (ORR) of compound 1 in combination therapy with ocitinib in patients with metastatic, RET fusion-positive non-small cell lung cancer progressing during or after ocitinib. ORR is defined as the proportion of patients that achieve a confirmed Complete Response (CR) or Partial Response (PR) according to RECIST 1.1.

Additional measures of anti-cancer activity were also evaluated in the study, including duration of response (DOR), Disease Control Rate (DCR), Clinical Benefit Rate (CBR), Progression Free Survival (PFS), and Overall Survival (OS). In addition, studies correlated compound 1PK parameters with safety endpoints and antitumor activity; further characterizing the safety and tolerability of compound 1 in combination with ocitinib; and assess the change in measures of quality of life (QoL) and symptom severity.

For additional measures, DOR is defined as the number of months from the time the criteria for CR or PR are first met to the first date that patients with confirmed CR or PR are objectively documented for Progressive Disease (PD); DCR is defined as experiencing Stable Disease (SD), Partial Response (PR) or Complete Response (CR) according to RECIST 1.1; a proportion of patients responding optimally according to either a Partial Response (PR) or a Complete Response (CR) of RECIST 1.1; and PFS is defined as the number of months from the first dose of study treatment to the earlier of PD or death due to any cause.

PK parameters for compound 1 included: population-derived estimates, including maximum plasma drug concentration (C)_max) Area under the plasma concentration versus time curve from time 0 to 24 hours post dose (AUC)_0-24) Plasma drug concentration 24 hours after steady state administration (C)₂₄) (ii) a As well as the type, frequency, severity, time and relationship to study drug of any AE, Severe Adverse Events (SAE), vital sign changes, ECG and safety laboratory tests.

The study included a standard 3+3 dose escalation portion to identify the MTD and/or RP2D when compound 1 was given in combination with ocitinib, followed by an extension phase to assess ORR and other measures of clinical activity. All patients enrolled in the phase 1 study portion must begin oxcetin treatment at an approved starting dose of 80 mg/day. In the phase 2 study section, patients who were toxic under prior axitinib treatment may begin taking axitinib at a lower initial dose, if needed. Dose levels of compound 1 were evaluated in dose increments of 200mg, 300mg and 400 mg. Patients in the extension phase received RP2D as determined by dose escalation. The extension followed a two-stage design in which 10 patients were treated initially. If patients at or above this first stage of 2/10 develop objective tumor responses, then a second stage recruits an additional 23 patients for a total of 33 patients for extended treatment.

For study eligibility, RET fusion status was determined by local or central assessment using tumor or blood samples taken at (or after) disease progression with EGFR inhibitors.

Study therapeutic compound 1 and ocitinib were administered by daily oral administration on a 28 day cycle. Dose modification is according to specific criteria based on observed toxicity.

Patients may continue to receive study therapeutics until excluded due to toxicity, non-compliance, withdrawal consent, death, or study termination. Patients who experience RECIST 1.1-defined disease progression but continue to experience clinical benefit in the treatment investigator's opinion may continue study treatment with approval. If compound 1 is permanently discontinued, the patient is considered to have completed the study treatment period; and receive other anti-cancer treatments (including ocitinib, if appropriate) as follow-up treatments during the survival follow-up. Patients in need of permanent withdrawal of ocitinib may continue monotherapy with compound 1 after approval.

All study visits are intended to be conducted on an outpatient basis, but may be conducted on an inpatient basis as needed. Disease assessments were performed every 8 weeks for the first two years, and then every 12 weeks thereafter. After discontinuation of study treatment, patients with no progressive disease record continue to receive disease assessments until progressive disease, initiation of another anti-tumor therapy, death, or study termination is recorded. Tumor response was assessed according to RECIST 1.1. Patients were also contacted 30 days after discontinuation of study treatment to assess safety and continued survival follow-up until death or study termination.

The patient population included the following participants: the age is more than or equal to 18 years when signing an informed consent; (ii) suffers from metastatic EGFR mutant NSCLC that is pathologically confirmed, unequivocally diagnosed; has at least one RECIST 1.1 evaluable target lesion; for stage 1 only: disease progression with radiologic record during or after prior treatment with any second or third generation EGFR inhibitor TKI; for stage 2 only: disease progression with radiologic record during or after previous treatment with ocitinib; detection of oncogenic RET fusions by local or central testing of circulating tumor nucleic acids in tumor tissue or blood using samples as described above (for patients eligible for local determination of RET status, the patient must also agree to submit blood and tissue samples to retrospectively confirm RET status by central testing); would provide archival tumor tissue (if samples obtained during or after prior treatment with axitinib following disease progression are available) or would accept a pretreatment biopsy if no suitable archival tumor tissue is available, and the researcher would consider the pretreatment biopsy safe and medically viable (if performed after baseline imaging, the pretreatment biopsy is taken from a non-target lesion); and the Performance Status (PS) of the Eastern Cooperative Oncology Group (ECOG) is 0-1.

The patient population did not include the following participants: with any additional known major driver changes (except for original EGFR mutations and RET fusions), including but not limited to targetable mutations of ALK, ROS1, MET, and BRAF; interstitial Lung Disease (ILD) or interstitial pneumonia, any prior history of radiation pneumonitis requiring steroid therapy, or any evidence of clinically active ILD within 28 days prior to enrollment; having a Central Nervous System (CNS) metastasis or a primary CNS tumor associated with progressive nervous system symptoms or requiring an increased corticosteroid dose to control CNS disease (if the patient requires a corticosteroid to treat CNS disease, the dose must be stable within 2 weeks prior to C1D 1); any anti-PD-1/PDL-1/CTLA treatment was received within 6 months and any other anti-cancer treatment (including systemic and radiation) within 14 days or 5 half-lives (whichever is shorter) before the first dose of study drug (excluding the previous ocitinib which may continue uninterrupted without flushing); over 30Gy of pulmonary radiation therapy within the first 6 months of participation; QTcF >480 milliseconds, a medical history with QT interval prolongation syndrome or torsades de pointes or a family history with QT interval prolongation syndrome; or any of the following within 14 days prior to the first dose of study drug:

a. platelet count<75x10⁹/L；

b. Absolute Neutrophil Count (ANC)<1.0x10⁹/L；

c. Hemoglobin <9.0g/dL (red blood cell infusion and erythropoietin can be used to achieve at least 9.0g/dL, but must be administered at least 2 weeks before the first dose of study drug;

d. aspartate Aminotransferase (AST) or alanine Aminotransferase (ALT) >3 × upper normal limit (ULN) if liver metastasis is not present; (ii) >5 × ULN if liver metastasis is present;

f. estimated (Cockroft-Gault formula) or measured creatinine clearance <40 mL/min; or

g. Total serum phosphorus was >5.5 mg/dL.

The phase 1 up-dosing phase included sample sizes of up to 18 patients. The total number of participants participating in the dose escalation depends on the observed safety profile that determines the number of participants per dose group and the number of groups required to confirm the recommended phase 2 dose (RP 2D).

For the phase 2 extension, a Simon two-stage design (Simon, 1989) was used, with a sample size of 10 patients (30% of total sample size) for phase 1, assuming a null hypothesis response rate of 5% and a replacement response rate of 25% (where single-sided α is 0.025 and power is 90%). The cumulative sample size for

stages

1 and 2 was 33. In phase 1, if the response rate does not exceed 1/10 (10%), the test is stopped because invalid hypotheses cannot be rejected. Otherwise, the trial continues to stage 2. If there are at least 5 responders out of all 33 patients, the null hypothesis is rejected.

For 33 patients, a probability of > 95% of at least one AE occurring at a frequency of 10% was observed; the probability of at least one AE occurring at a frequency of 20% was observed to be > 99%.

For the analysis population, the Response Evaluable Population (REP) included all patients with measurable disease at baseline, receiving at least one dose of each study treatment (compound 1 and ocitinib), and having evaluable post-baseline tumor response assessments. REP is used for preliminary analysis of ORR, DCR and CBR. The safe population (including all patients receiving at least one dose of compound 1) was used for residual efficacy endpoints and safety.

For REP, the number and percentage of patients with objective responses and a bilateral 95% confidence interval are given using the exact cloner Pearson method. Based on the same approach, DCR and CBR and a bilateral 95% confidence interval were estimated. For patients who achieved objective responses, DOR was calculated from the time the CR/PR criteria were first met to the first date objectively documented to progressive disease. Responders who did not experience a recorded progressive disease or death were reviewed at the time of the last response assessment, and the median and 95% CI thereof were estimated using the Kaplan-Meier method.

Progression-free survival was analyzed using the Kaplan-Meier method. If the patient does not experience progressive disease or death, the patient is reviewed at the last response assessment.

Safety analysis included data summaries of clinical and laboratory parameters as well as AEs. Based on NCI CTCAE v 5.0, the number and percentage of patients experiencing one or more AEs were summarized by relationship and severity to study drug. Adverse events are encoded using the supervised active medical dictionary (MedDRA). Laboratory parameters were summarized using descriptive statistics, by post-treatment changes from baseline, and a list of data with clinically significant abnormalities. The vital signs and ECG data are summarized using descriptive statistics. Compound 1 plasma concentration data were tabulated using descriptive statistics. The exposure parameters of compound 1 correlated with safety endpoints and antitumor activity.

Claims

2. The method of claim 1, wherein the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof.

3. The method of claim 1, wherein the at least one RET inhibitor is selected from the group consisting of: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multiverinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, TPX-0046, SL-1001, Nintenib, Ponatinib, Serratinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

4. The method of claim 1, wherein the at least one RET inhibitor is a selective RET inhibitor.

5. The method of any one of claims 1 to 4, wherein the at least one EGFR inhibitor is a selective EGFR inhibitor.

6. The method of any one of claims 1 to 4, wherein the at least one EGFR inhibitor is a third-generation EGFR inhibitor.

7. The method of any one of claims 1 to 4, wherein the at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof.

8. The method of any one of claims 1 to 7, wherein the EGFR mutant cancer is characterized by at least one EGFR mutation selected from T790M, C797S, and L792H.

9. The method of any one of claims 1 to 8, wherein the EGFR mutant cancer is further characterized by at least one RET fusion.

10. The method of claim 9, wherein the EGFR mutant cancer is further characterized by a CCDC6-RET fusion.

11. The method of any one of claims 1 to 10, wherein the EGFR mutant cancer is lung cancer.

12. The method of claim 11, wherein the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.

13. The method of any one of claims 1 to 12, wherein the patient is a human.

14. The method of any one of claims 1 to 13, wherein the patient was previously treated with at least one EGFR inhibitor.

15. The method of any one of claims 1 to 14, wherein the patient gains resistance to at least one EGFR inhibitor.

16. The method of any one of claims 1, 2, and 4 to 15, wherein:

the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof;

the at least one RET inhibitor is orally administered to the patient once daily; and is

17. The method of claim 16, wherein the therapeutically effective amount of the at least one RET inhibitor is 200mg to 300mg of compound 1 or a weight equivalent of a pharmaceutically acceptable salt thereof.

18. The method of any one of claims 7 to 17, wherein

The at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof;

the at least one EGFR inhibitor is orally administered to the patient once daily; and is

The therapeutically effective amount of the at least one EGFR inhibitor is 80mg of ocitinib or a weight equivalent of a pharmaceutically acceptable salt thereof.

20. The combination therapy of claim 19, wherein the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof.

21. The combination therapy of claim 19, wherein the at least one RET inhibitor is selected from the group consisting of: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multiverinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, TPX-0046, SL-1001, Nintenib, Ponatinib, Serratinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

22. The combination therapy of claim 19, wherein the at least one RET inhibitor is a selective RET inhibitor.

23. The combination therapy of any one of claims 19 to 22, wherein the at least one EGFR inhibitor is a selective EGFR inhibitor.

24. The combination therapy of any one of claims 19 to 22, wherein the at least one EGFR inhibitor is a third-generation EGFR inhibitor.

25. The combination therapy of claim 20, wherein compound 1 is present in an amount of 200mg to 400 mg.

26. The combination therapy of claim 20, wherein compound 1 is present in an amount of 200mg to 300 mg.

27. The combination therapy of any one of claims 19 to 22, 25 or 26, wherein the at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof.

28. The combination therapy of claim 27, wherein ocitinib is present in an amount of 80 mg.

(a) obtaining a biological sample from the patient;

(b) detecting the presence or absence of at least one RET fusion in the biological sample; and

30. The method of claim 29, wherein the at least one RET inhibitor is selected from compound 1 and pharmaceutically acceptable salts thereof.

31. The method of claim 29, wherein the at least one RET inhibitor is selected from the group consisting of: alaninib, Apatinib, BOS172738(DS-5010), cabozantinib (XL184), Multiverinib (TKI258), GSK3179106, GSK3352589, lenvatinib, LOXO-292, TPX-0046, SL-1001, Nintenib, Ponatinib, Serratinib (MGCD516), sorafenib, sunitinib, regorafenib (BAY 73-4506), RXDX-105, vandetanib, XL999, and pharmaceutically acceptable salts of any of the foregoing.

32. The method of claim 29, wherein the at least one RET inhibitor is a selective RET inhibitor.

33. The method of any one of claims 29 to 32, wherein the at least one EGFR inhibitor is selected from ocitinib and pharmaceutically acceptable salts thereof.

34. The method of any one of claims 29 to 32, wherein the at least one EGFR inhibitor is a selective EGFR inhibitor.

35. The method of any one of claims 29 to 32, wherein the at least one EGFR inhibitor is a third-generation EGFR inhibitor.

36. The method of any one of claims 29 to 35, wherein the EGFR mutant cancer is characterized by at least one EGFR mutation selected from T790M, C797S, and L792H.

37. The method of any one of claims 29 to 36, wherein the at least one RET fusion is a CCDC6-RET fusion.

38. The method of any one of claims 29 to 37, wherein the EGFR mutant cancer is lung cancer.

39. The method of claim 38, wherein the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.

40. The method of any one of claims 29 to 39, wherein the patient is a human.

41. The method of any one of claims 29 to 40, wherein the patient was previously treated with at least one EGFR inhibitor.

42. The method of any one of claims 29 to 41, wherein the patient gains resistance to at least one EGFR inhibitor.